The present invention relates to a method for analyzing DNA information in the fields of clinical diagnosis and life science, and a device therefor.
In the rapidly progressing technology of DNA analysis, recently, significant attention has been focused on the analysis of information of DNA/RNA sequence in the fields of clinical diagnosis and life science. In the field of life science, for example, advances have been made for determining the entire nucleotide sequence of a variety of animal and plant DNAs, as illustrated by the Human Genome Project. Thus, the coding region of a novel protein and the regulatory site of the expression thereof have been analyzed gradually, involving also the elucidation of pathogenic genes such as oncogenes and the like.
In the field of clinical diagnosis, alternatively, the introduction of the technology of DNA analysis has been accelerated toward the identification of a variety of etiology and laboratory tests, on the basis of the fruitful results of these research works. The diagnosis of infectious diseases including viral hepatitis type C and AIDS (acquired immunodeficiency syndrome) due to HIV (human immunodeficiency virus) infection is one example of the fields for which the introduction of DNA diagnosis has been highly desired because of the high detection sensitivity required therefor and because of the relation between the infectious performance of these viruses (retroviruses) and the DNA/RNA polymorphism. For the laboratory tests of tumor cells, which are now dependent on empirical pathological diagnosis, and for the tests of the HLA (human leukocyte antigen) type with the sample number rapidly increasing from the demand of the registration at the myeloid bank, the introduction of the technology of DNA analysis has been desired to give accurate and precise information.
Great progress has been made recently in the DNA analysis technology desired in such fields, wherein a method for separating a slight difference in DNA sequence utilizing the difference in the conformation of a single-stranded DNA during electrophoresis has been developed, in addition to the conventional DNA sequencing and hybridization methods. As introduced in Genomics, Vol. 5, pp. 874-879, 1989, for example, the method designated as SSCP (Single Strand Conformation Polymorphisms) has been drawing attention as a technique for detecting even a single base substitution at a high sensitivity. The separating method detects the difference in sequence by detecting the difference in the conformation as the difference in the mobility on gel electrophoresis, with attention focused on the finding that leaving DNA, normally composed of a pair of complementary double strands, in a single strand state, typically, the single-stranded DNA autonomously associates by itself within the molecule under appropriate conditions (ion strength, temperature and the like) and forms a certain conformation specific to the sequence, which conformation varies depending on the sequence.
The method detects the difference in DNA at a high sensitivity, but because electrophoresis is employed in the process of separation, such a long period of time is required for the separation that a high throughput is realized only with much difficulty. The selection of the conditions for efficiently reflecting the difference in DNA sequence over the difference in the mobility on electrophoresis is difficult. Still furthermore, the method is hardly automated, and additionally, the method involves another drawback in requiring the separation in some case under a plurality of conditions so as to thoroughly separate the entire polymorphism.
A method called denaturant gradient gel electrophoresis for detecting a slight difference in DNA sequence has also been proposed, but because the method also employs electrophoresis, it has the same drawbacks as described above.
It has been known that the difference between the denaturing conditions of a double-stranded DNA with a completely complementary sequence and the conditions of a double-stranded DNA with an almost complementary but not completely complementary sequence can be detected as the difference in absorbance change when such double-stranded DNA is denatured into a single-stranded DNA (melting curve) (for example, see I. V. Razlutuskii, L. S. Shlyakhtenko and Yu. L. lyubchenko: Nucleic Acids Research, Vol. 15, No. 16, pp. 6665-6676 (1987)). Furthermore, it has been known that the type of a single-stranded RNA forming conformation (hair pin, stem, loop structure, etc.) can be detected and identified by the change in the absorbance when the base pairing of the single-stranded RNA is denatured (melting curve) (for example, see L. G. Laing and D. E. Draper: J. Mol. Biol. (1994) 237, 560-576).
According to these methods, an electrophoresis procedure is not required after a sample is collected, so that these methods are advantageous in that the procedures are simple and the measurements are easily carried out as an optical measurement, with higher reliability.
Additionally, a technique has been proposed, comprising optically measuring and detecting the phenomenon that when a double-stranded DNA having a completely complementary sequence and a double-stranded DNA having a nearly completely but not completely complementary sequence are denatured, the fluorescent energy transfer induced by two types of fluorophors individually labeling each of the complementary strands is eliminated, thereby detecting the difference in the sequences of the two types of DNAs which are not completely complementary (Japanese Patent Laid-open No. Hei 7-31500). Because no electrophoresis procedure is then required after a sample is collected, the same advantage is achieved as described in the aforementioned example.
However, it is only sequence compositions (GC contents, etc.) or deletion/insertion of bases that these methods can detect. The identification of detailed differences in sequence, particularly DNA polymorphism including single-base substitution, is substantially difficult by using these methods. These methods require the regulation of denaturing conditions to be carried out at such an extremely low rate that the methods have not been able to achieve the detection and determination at a high throughput.
Furthermore, no examination has been made about direct optical analysis of DNA polymorphism including single-base substitution in a single-stranded DNA.